Antipathogenic devices and methods thereof

ABSTRACT

Disclosed herein are compositions, devices and methods for inactivating viruses, bacteria, and fungi. The compositions, methods, and devices may include coatings or slurries such as silicon nitride powder coatings or slurries for the inactivation of viruses, bacteria, and/or fungi.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of Ser. No. 17/521,270,filed Nov. 8, 2021, which is a divisional application of Ser. No.16/550,605, filed Aug. 26, 2019 now U.S. Pat. No. 11,192,787, whichclaims the benefit of U.S. Provisional Application No. 62/727,724, filedSep. 6, 2018, and U.S. Provisional Application No. 62/800,034, filedFeb. 1, 2019, the contents of which are entirely incorporated byreference herein.

FIELD

The present disclosure relates to antiviral, antibacterial, andantifungal composition, systems, methods, and devices. Morespecifically, the disclosure relates to silicon nitride compositions,devices, and coatings for the inactivation and lysis of viruses,bacteria, and fungi.

BACKGROUND

The need for safe and reliable inactivation, removal, or lysis ofviruses, bacteria, and fungi is universal. There is a broad need tocontrol the pathogens that affect human health and agriculturalproducts. Not only is there a need for materials that possessantipathogenic properties for human medicinal therapies, but also foruse as surface coatings and/or composites for various medical devices orequipment, examination tables, clothing, filters, masks, gloves,catheters, endoscopic instruments, and the like.

Furthermore, application of antipathogenic materials is greatly neededin agriculture. Up to 15% of the world's edible annual crops aredestroyed due to their susceptibility to plant-based viruses, bacteria,and fungi. For example, Plasmopara viticola is considered to be one ofthe most devastating diseases of grape vines in climates with relativelywarm and humid summers and has markedly reduced crop yields in France,Spain, and Italy. Furthermore, there is increasing concern that themycotoxins produced by these fungi have an overall negative impact onhuman health and longevity. Conventional pharmaceutical pathogeninactivation methods include the use of specially engineered organicpetrochemicals, antibiotics, genetic engineering, or through the use ofsolid-state inactivators (e.g., cuprous oxide, Cu₂O, and silver nitrate,AgNO₃). While these therapies are quite effective, there are significantenvironmental health and safety concerns with their use. Newpetrochemical compounds may have chronic residual effects to humans,wildlife, plants, and soil. Extensive use of antibiotics in humans,animals and on agricultural crops increases the inherent resistance ofbacterial pathogens. Genetic engineering of crops to resist disease isincreasingly unpopular and politically unpalatable. Solid-stateinactivators release Cu and Ag ions which might induce damage tomammalian cells. In addition, each of these approaches to the control ofpathogens has come under increased regulatory scrutiny.

Therefore, there is a need for safe and reliable methods to inactivateand kill viruses, bacteria, and fungi that may be applied to medicaldevices, equipment, clothing, or other systems which may have prolongedcontact with the human body or be used in various agriculturalapplications to treat viral or bacterial diseases and fungal infections.

SUMMARY

Provided herein is a device having silicon nitride on at least a portionof a surface of the device, wherein the silicon nitride is present in aconcentration sufficient to inactivate a pathogen on the surface of thedevice. The device may include a silicon nitride coating. The siliconnitride may present in a concentration of about 1 wt. % to about 100 wt.%, for example 15 wt. % silicon nitride. Also provided herein is amethod of treating or preventing a pathogen at a location in a humanpatient. The method may include contacting the patient with a devicecomprising silicon nitride. In another aspect, a method of inactivatinga pathogen may include contacting an apparatus comprising siliconnitride at a concentration of about 1 wt. % to about 100 wt. % with thevirus. The methods may further include coating the apparatus with asilicon nitride powder on the surface of the apparatus and/orincorporating a silicon nitride powder within the apparatus. The siliconnitride in the device, apparatus, and/or coating may be present in aconcentration sufficient to inactivate the pathogen. The apparatus maybe in contact with a patient for as long as needed to inactivate thepathogen. For example, the apparatus may be in contact with the patientfor at least 1 minute or may be permanently implanted within thepatient.

The silicon nitride in the device or apparatus may be present in theform of a powder. In an aspect, the pathogen may be Influenza A. Thesilicon nitride may decrease viral action by alkalinetransesterification and reduce the activity of hemagglutinin.

Further provided herein is a composition for inactivating a pathogen mayinclude silicon nitride in a concentration of about 1 vol. % to about 30vol. %, for example about 1.5 vol % silicon nitride. In another aspect,a method of inactivating a pathogen may include contacting a compositioncomprising silicon nitride at a concentration of about 1 vol. % to about30 vol. % with the pathogen. The method may further include spraying thecomposition onto the surface of a plant to contact the pathogen. Thecomposition may be in contact with the pathogen for at least 1 minute.The composition may include a slurry of silicon nitride particles andwater.

The silicon nitride may be present in a concentration sufficient toinactivate the pathogen. The silicon nitride particles may attach tospores of the pathogen. The pathogen may be Plasmopara viticola. Theplant may be Cabernet Sauvignon or Cannonau.

Further provided herein is a method of treating or preventing a pathogenat a location in on a plant. The method may include contacting the plantwith a slurry comprising silicon nitride. The slurry may include about 1vol. % to about 30 vol. % silicon nitride. The silicon nitride may bepresent in the in a concentration sufficient to inactivate the pathogen.In some aspects, the pathogen is Plasmopara viticola, and the plant isCabernet Sauvignon or Cannonau. The composition may be in contact withthe pathogen for at least 1 minute.

Other aspects and iterations of the invention are described morethoroughly below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of the Influenza A virus.

FIG. 2A is an illustration of a virus exposed to 0 wt. %, 7.5 wt. %, 15wt. %, and 30 wt. % Si₃N₄ for 10 minutes.

FIG. 2B is an illustration of methods used to determine viability ofcells inoculated with a virus exposed to Si₃N₄ according to FIG. 2A.

FIG. 3A is an illustration of a virus exposed to 15 wt. % Si₃N₄ for 1,5, 10, and 30 minutes.

FIG. 3B is an illustration of methods used to determine viability of avirus after exposure to Si₃N₄ according to FIG. 3A.

FIG. 4A is a graph of PFU/100 μl for Influenza A exposed to 0 wt. %, 7.5wt. %, 15 wt. %, and 30 wt. % Si₃N₄ for 10 minutes according to FIG. 2A.

FIG. 4B is a graph of cell survivability of cells inoculated withInfluenza A exposed to 7.5 wt. %, 15 wt. %, and 30 wt. % Si₃N₄ for 10minutes according to FIG. 2B.

FIG. 5 includes photographs of cells inoculated with different ratios ofvirus to slurry that had been exposed to various concentrations ofSi₃N₄.

FIG. 6A shows a fluorescence microscopy image of MDCK cells beforeinoculation.

FIG. 6B shows a fluorescence microscopy image of MDCK cells afterinoculation with a virus exposed to the control.

FIG. 6C shows a fluorescence microscopy image of MDCK cells afterinoculation with a virus exposed to 30 wt. % Si₃N₄.

FIG. 7A is a graph of PFU/100 μl for Influenza A exposed to 15 wt. %Si₃N₄ for 1 minute, 5 minutes, 10 minutes, or 30 minutes at roomtemperature.

FIG. 7B is a graph of cell survivability of cells inoculated withInfluenza A exposed to 15 wt. % Si₃N₄ for 1 minute, 5 minutes, 10minutes, or 30 minutes at room temperature.

FIG. 8A is a graph of PFU/100 μl for Influenza A exposed to 15 wt. %Si₃N₄ for 1 minute, 5 minutes, 10 minutes, or 30 minutes at 4° C.

FIG. 8B is a graph of cell survivability of cells inoculated withInfluenza A exposed to 15 wt. % Si₃N₄ for 1 minute, 5 minutes, 10minutes, or 30 minutes at 4° C.

FIG. 9A shows the Raman spectrum of Influenza A virus beforeinactivation.

FIG. 9B shows changes in the Raman spectrum of the Influenza A virusrelevant to chemical modifications in RNA and hemagglutinin afterinactivation after 1 minute of exposure.

FIG. 10 shows NH₃ inactivates Influenza A virus by the mechanism ofalkaline transesterification.

FIG. 11 shows O—P—O stretching in pentacoordinate phosphate group afterinactivation.

FIG. 12A shows vibrational modes of methionine in the hemagglutininstructure.

FIG. 12B shows methionine's structural change in the presence ofammonia.

FIG. 13 shows C—S stretching methionine to homocysteine afterinactivation.

FIG. 14A is a graph of PFU/100 μl for Feline calicivirus exposed to 15wt. % or 30 wt. % Si₃N₄ for 1 minute, 10 minutes, or 30 minutes.

FIG. 14B is a graph of cell survivability of cells inoculated withFeline calicivirus exposed to 30 wt. % Si₃N₄ for 1 minute, 10 minutes,30 minutes, or 60 minutes.

FIG. 15A shows the H1 H1 Influenza A virus (nucleoprotein, NP) stainedred after 10 minutes of exposure to a slurry of 15 wt. % silicon nitrideand after its inoculation into a biogenic medium containing MDCK cellsstained green for the presence of filamentous actin (F-actin) proteins.

FIG. 15B shows the NP stained H1H1 Influenza A virus from FIG. 15A.

FIG. 15C shows the F-actin stained MDCK cells from FIG. 15A.

FIG. 16A shows the H1 H1 Influenza A virus (nucleoprotein, NP) stainedred without exposure to silicon nitride and after its inoculation into abiogenic medium containing MDCK cells stained green for the presence offilamentous actin (F-actin) proteins.

FIG. 16B shows the NP stained H1H1 Influenza A virus from FIG. 16A.

FIG. 16C shows the F-actin stained MDCK cells from FIG. 16A.

FIG. 17A shows Cabernet Sauvignon leaves inoculated with Plasmoparaviticola untreated.

FIG. 17B shows Cabernet Sauvignon leaves inoculated with Plasmoparaviticola treated for 1 minute with 1.5 vol. % Si₃N₄ powder.

FIG. 18A shows untreated spore sacs.

FIG. 18B shows spore sacs in the presence of Si₃N₄.

FIG. 19 is a graph of the infected leaf area of Cabernet Sauvignon andCannonau leaves with control and treated Plasmopara viticola.

DETAILED DESCRIPTION

Various embodiments of the disclosure are discussed in detail below.While specific implementations are discussed, it should be understoodthat this is done for illustration purposes only. A person skilled inthe relevant art will recognize that other components and configurationsmay be used without parting from the spirit and scope of the disclosure.Thus, the following description and drawings are illustrative and arenot to be construed as limiting. Numerous specific details are describedto provide a thorough understanding of the disclosure. However, incertain instances, well-known or conventional details are not describedin order to avoid obscuring the description. References to one or anembodiment in the present disclosure can be references to the sameembodiment or any embodiment; and, such references mean at least one ofthe embodiments.

Reference to “one embodiment” or “an embodiment” means that a particularfeature, structure, or characteristic described in connection with theembodiment is included in at least one embodiment of the disclosure. Theappearances of the phrase “in one embodiment” in various places in thespecification are not necessarily all referring to the same embodiment,nor are separate or alternative embodiments mutually exclusive of otherembodiments. Moreover, various features are described which may beexhibited by some embodiments and not by others.

The term “apparatus” as used herein includes compositions, devices,surface coatings, and/or composites. In some examples the apparatus mayinclude various medical devices or equipment, examination tables,clothing, filters, masks, gloves, catheters, endoscopic instruments, andthe like. The apparatus may be metallic, polymeric, and/or ceramic (ex.silicon nitride and/or other ceramic materials).

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the disclosure, and in thespecific context where each term is used. Alternative language andsynonyms may be used for any one or more of the terms discussed herein,and no special significance should be placed upon whether or not a termis elaborated or discussed herein. In some cases, synonyms for certainterms are provided. A recital of one or more synonyms does not excludethe use of other synonyms. The use of examples anywhere in thisspecification including examples of any terms discussed herein isillustrative only, and is not intended to further limit the scope andmeaning of the disclosure or of any example term. Likewise, thedisclosure is not limited to various embodiments given in thisspecification.

Additional features and advantages of the disclosure will be set forthin the description which follows, and in part will be obvious from thedescription, or can be learned by practice of the herein disclosedprinciples. The features and advantages of the disclosure can berealized and obtained by means of the instruments and combinationsparticularly pointed out in the appended claims. These and otherfeatures of the disclosure will become more fully apparent from thefollowing description and appended claims, or can be learned by thepractice of the principles set forth herein.

Provided herein are antipathogenic devices, compositions, andapparatuses that include silicon nitride (Si₃N₄) for the inactivation ofviruses, bacteria, and fungi. Silicon nitride possesses a unique surfacechemistry which is biocompatible and provides a number of biomedicalapplications including 1) concurrent osteogenesis, osteoinduction,osteoconduction, and bacteriostasis, such as in spinal and dentalimplants; 2) killing of both gram-positive and gram-negative bacteriaaccording to different mechanisms; 3) inactivation of human and animalviruses, bacteria, and fungi as well as plant-based viruses, bacteria,and fungi; and 4) polymer- or metal-matrix composites, natural ormanmade fibers, polymers, or metals containing silicon nitride powderretain key silicon nitride bone restorative, bacteriostatic, antiviral,and antifungal properties.

In an embodiment, an antipathogenic composition may include siliconnitride. For example, the antipathogenic composition may include siliconnitride powder. In some embodiments, the antipathogenic composition maybe a monolithic component comprising 100% silicon nitride. Such acomponent can be fully dense possessing no internal porosity, or it maybe porous, having a porosity that ranges from about 1% to about 80%. Themonolithic component may be used as a medical device or may be used inan apparatus in which the inactivation of a virus, bacteria, and/orfungi may be desired. In another embodiment, antipathogenic compositionmay be incorporated within a device or in a coating to inactivateviruses, bacteria, and fungi. In some embodiments, the antipathogeniccomposition may be a slurry comprising silicon nitride powder. Forexample, the antipathogenic composition may be sprayed onto the surfaceof plants for the inactivation of agricultural pathogens.

In some embodiments, the antipathogenic composition may inactivate humanviruses, bacteria, and/or fungi. Non-limiting examples of viruses thatmay be inactivated by the antipathogenic composition include Influenza Aand Feline calicivirus. For example, a silicon nitride bioceramic may beeffective in the inactivation of the Influenza A virus. In someembodiments, a silicon nitride coating may decrease antibacterial andantiviral resistance and/or promote bone tissue restoration. In someembodiments, the antipathogenic composition may inactivate agriculturalviruses, bacteria, and/or fungi. Non-limiting examples of agriculturalfungi that may be inactivated by the antipathogenic composition includePlasmopara viticola (downy mildew) or similar plant pathogens.

Without being limited to a particular theory, silicon nitride mayprovide a surface chemistry such that ammonia (NH₃) is available forvirus, bacteria, or fungi inactivation. The surface chemistry of siliconnitride may be shown as follows:

Si₃N₄+6H₂O→3SiO₂+4NH₃

SiO₂+2H₂O→Si(OH)₄

Nitrogen elutes faster (within minutes) than silicon because surfacesilanols are relatively stable. For viruses, it was surprisingly foundthat silicon nitride may provide for RNA cleavage via alkalinetransesterification which leads to loss in genome integrity and virusinactivation. This may also reduce the activity of hemagglutinin.

In an embodiment, the antipathogenic composition may exhibit elutionkinetics that show: (i) a slow but continuous elution of ammonia fromthe solid state rather than from the usual gas state; (ii) no damage ornegative effect to cells; and (iii) an intelligent elution increasingwith decreasing pH. The inorganic nature of silicon nitride may be morebeneficial than the use of petrochemical or organometallic fungicideswhich are known to have residual effects in soil, on plants, and intheir fruit.

A device or apparatus may include silicon nitride on at least a portionof a surface of the device for antiviral, antibacterial, or antifungalaction. In an embodiment, a device may include a silicon nitride coatingon at least a portion of a surface of the device. The silicon nitridecoating may be applied to the surface of the device as a powder. In someembodiments, the powder may be micrometric in size. In otherembodiments, the silicon nitride may be incorporated into the device.For example, a device may incorporate silicon nitride powder within thebody of the device. In one embodiment, the device may be made of siliconnitride.

The silicon nitride coating may be present on the surface of a device ina concentration of about 1 wt. % to about 100 wt. %. In variousembodiments, the coating may include about 1 wt. %, 2 wt. %, 5 wt. %,7.5 wt. %, 8.3 wt. %, 10 wt. %, 15 wt. %, 16.7 wt. %, 20 wt. %, 25 wt.%, 30 wt. %, 33.3 wt. %, 35 wt. %, or 40 wt. % silicon nitride powder.In at least one example, the coating includes about 15 wt. % siliconnitride. In some embodiments, silicon nitride may be present in or onthe surface of a device or apparatus in a concentration of about 1 wt. %to about 100 wt. %. In various embodiments, a device or apparatus mayinclude about 1 wt. %, 2 wt. %, 5 wt. %, 7.5 wt. %, 8.3 wt. %, 10 wt. %,15 wt. %, 16.7 wt. %, 20 wt. %, 25 wt. %, 30 wt. %, 33.3 wt. %, 35 wt.%, 40 wt. %, 50 wt. %, 60 wt. %, 60 wt. %, 70 wt %, 80 wt. %, 90 wt. %,to 100 wt. % silicon nitride.

In various embodiments, a device or apparatus that includes siliconnitride for antipathogenic properties may be a medical device.Non-limiting examples of devices or apparatuses include orthopedicimplants, spinal implants, pedicle screws, dental implants, in-dwellingcatheters, endotracheal tubes, colonoscopy scopes, and other similardevices.

In some embodiments, silicon nitride may be incorporated within orapplied as a coating to materials or apparatuses for antipathogenicproperties such as polymers and fabrics, surgical gowns, tubing,clothing, air and water filters, masks, tables such as hospital exam andsurgical tables, desks, toys, filters such as air conditioner filters,or toothbrushes.

In other embodiments, silicon nitride powder may be incorporated intocompositions including, but not limited to slurries, suspensions, gels,sprays, or toothpaste. In other embodiments, silicon nitride may bemixed with water along with any appropriate dispersants and slurrystabilization agents, and thereafter applied by spraying the slurry ontovarious agricultural plants, fruit-trees, vines, grain crops, and thelike. For example, a silicon nitride slurry may be sprayed on fungiinfected grape leaves.

In an example, the antipathogenic composition may be a slurry of siliconnitride powder and water. The silicon nitride powder may be present inthe slurry in a concentration of about 0.1 vol. % to about 20 vol. %. Invarious embodiments, the slurry may include about 0.1 vol. %, 0.5 vol.%, 1 vol. %, 1.5 vol. %, 2 vol. %, 5 vol. %, 10 vol. %, 15 vol. %, or 20vol. % silicon nitride.

Further provided herein is a method of inactivating a pathogen bycontacting a virus, bacteria, and/or fungus with an antipathogeniccomposition comprising silicon nitride. In an embodiment, the method mayinclude coating a device or apparatus with silicon nitride andcontacting the coated apparatus with the virus, bacterium, or fungus.Coating the apparatus may include applying a silicon nitride powder to asurface of the apparatus. In other embodiments, the silicon nitridepowder may be incorporated within the device or apparatus.

In further embodiments, the method may include contacting a siliconnitride slurry with the surface of living agricultural plants, trees,grains, etc. infected with a plant-based pathogen. In an embodiment,infected leaves may be sprayed with an about 1 vol. % to about 40 vol %slurry of silicon nitride in water. The leaves may be exposed to thesilicon nitride slurry for at least 1 minute, at least 5 minutes, atleast 10 minutes, at least 20 minutes, at least 30 minutes, at least 1hour, at least 2 hours, at least 5 hours, or at least 1 day. In variousexamples, the infected area of leaves may be reduced by at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, or atleast 99%. In an example, after 1 minute of exposure, the infected areaof the leaves may be reduced by about 95%.

Without being limited to a particular theory, the antipathogeniccomposition may decrease viral action by alkaline transesterificationand reduce the activity of hemagglutinin. It was surprisingly found thatsilicon nitride powder (i) remarkably decreases viral action by alkalinetransesterification through the breakage of RNA internucleotide linkagesand (ii) markedly reduced the activity of hemagglutinin thus disruptinghost cell recognition by denaturing protein structures on viral surfacesleading to the inactivation of viruses regardless of the presence of aviral envelope.

In an embodiment, the antipathogenic composition may exhibit elutionkinetics that show: (i) a slow but continuous elution of ammonia fromthe solid state rather than from the usual gas state; (ii) no damage ornegative effect to cells; and (iii) an intelligent elution increasingwith decreasing pH. Moreover, the inorganic nature of silicon nitridemay be more beneficial than the use of petrochemical or organometallicfungicides which are known to have residual effects in soil, on plants,and in their fruit.

It was also surprisingly found that silicon nitride particles may beelectrically attracted to and attach to the spores of the pathogen.

Also provided herein is a method of treating or preventing a pathogen ata location in a human patient. For example, the pathogen may be a virus,bacterium, or fungus. The method may include contacting the patient witha device, apparatus, or composition comprising silicon nitride. Withoutbeing limited to any one theory, the silicon nitride inactivates thevirus (for example, Influenza A), bacterium, or fungus. The device,apparatus, or composition may include about 1 wt. % to about 100 wt. %silicon nitride. In some examples, the device or apparatus may includeabout 1 wt. % to about 100 wt. % silicon nitride on the surface of thedevice or apparatus. In an embodiment, the device or apparatus may be amonolithic silicon nitride ceramic. In another embodiment, the device orapparatus may include a silicon nitride coating, such as a siliconnitride powder coating. In another embodiment, the device or apparatusmay incorporate silicon nitride into the body of the device. Forexample, silicon nitride powder may be ground in or otherwiseincorporated into the body of the device or apparatus using methodsknown in the art.

In some embodiments, the device or apparatus may be contacted with thepatient for at least 1 minute, at least 5 minutes, at least 30 minutes,at least 1 hour, at least 2 hours, at least 5 hours, or at least 1 day.In at least one example, the device or apparatus may be permanentlyimplanted in the patient.

Also provided herein is a method of treating or preventing a pathogen ata location in a plant. For example, the pathogen may be a virus,bacterium, or fungus. The method may include contacting the plant with acomposition comprising silicon nitride. Without being limited to any onetheory, the silicon nitride inactivates the virus, bacterium, or fungus(for example, Plasmopara viticola). In some embodiments, the compositionmay include a silicon nitride slurry in water containing up to 40 vol. %silicon nitride with appropriate dispersants and slurry stabilizationagents. The composition may be applied to living agricultural plants,trees, grains and the like to inactivate and kill or prevent the growthof viruses, bacteria, and fungi after being in contact with them for atleast 1 minute, at least 5 minutes, at least 30 minutes, at least 1hour, at least 2 hours, at least 5 hours, or at least 1 day.

Examples Example 1: Effect of Silicon Nitride Concentration on VirusInactivation

To show the effect of silicon nitride concentration on the inactivationof viruses, Influenza A was exposed to various concentrations of Si₃N₄powder. To prepare the silicon nitride, a specific weight of siliconnitride powder mixed with pure distilled water. For instance, 7.5 g ofsilicon nitride was dispersed in 92.5 g of pure distilled water. Thevirus was added to this mixture in concentrations of 1:1, 1:10 and 1:100virus/mixture, respectively. These mixtures were then allowed toincubate under gentle agitation for 10 minutes at 4° C. Influenza A wasexposed to 0 wt. %, 7.5 wt. %, 15 wt. %, and 30 wt. % Si₃N₄ for 10minutes at 4° C., as illustrated in FIG. 2A. The mixtures were thenfiltered to remove the silicon nitride powder.

Influenza A virus-inoculated Madin-Darby canine kidney (MDCK) cells werethen observed for the effectiveness of Si₃N₄ in inactivating theInfluenza A. The remaining mixtures were then inoculated into Petridishes containing living MDCK cells within a biogenic medium. The amountof living MDCK cells were subsequently counted using staining methodsafter 3 days exposure. The viability of MDCK cells was determined afterinoculating the cells for 3 days with Influenza A exposed to Si₃N₄according to FIG. 2B.

FIG. 4A is a graph of PFU/100 μl for Influenza A exposed to 0 wt. %, 7.5wt. %, 15 wt. %, and 30 wt. % Si₃N₄ for 10 minutes. FIG. 4B is a graphof cell survivability of cells inoculated with Influenza A exposed to7.5 wt. %, 15 wt. %, and 30 wt. % Si₃N₄ for 10 minutes.

Example 2: Effect of Exposure Time and Temperature on Virus Inactivation

To show the effect of silicon nitride on the inactivation of viruses,Influenza A was exposed to a fixed concentration of Si₃N₄ powder (15 wt.%) for various times and temperatures. The mixture was then allowed toincubate under gentle agitation for 1-30 minutes at room temperature andat 4° C. For example, Influenza A was exposed to 15 wt. % Si₃N₄ for 1,5, 10, or 30 minutes at room temperature or 4° C., as illustrated inFIG. 3A. Influenza A virus-inoculated Madin-Darby canine kidney (MDCK)cells were then observed for the effectiveness of Si₃N₄ in inactivatingthe Influenza A. The viability of MDCK cells was determined afterinoculating the cells for 3 days with Influenza A exposed to Si₃N₄according to FIG. 3B.

FIG. 7A is a graph of PFU/100 μl for Influenza A exposed to 15 wt. %Si₃N₄ for 1 minute, 5 minutes, 10 minutes, or 30 minutes at roomtemperature. FIG. 7B is a graph of cell survivability of cellsinoculated with Influenza A exposed to 15 wt. % Si₃N₄ for 1 minute, 5minutes, 10 minutes, or 30 minutes at room temperature.

FIG. 8A is a graph of PFU/100 μl for Influenza A exposed to 15 wt. %Si₃N₄ for 1 minute, 5 minutes, 10 minutes, or 30 minutes at 4° C. FIG.8B is a graph of cell survivability of cells inoculated with Influenza Aexposed to 15 wt. % Si₃N₄ for 1 minute, 5 minutes, 10 minutes, or 30minutes at 4° C.

Example 3: Effect of Silicon Nitride on H1H1 Influenza a Inactivation

To show the effect of silicon nitride on the inactivation of viruses,Influenza A was exposed to a slurry of 15 wt. % silicon nitride for 10minutes.

FIGS. 15A-15C show the H1H1 Influenza A virus (A/Puerto Rico/8/1934 H1N₁ (PR8)) stained red (nucleoprotein, NP) after its inoculation into abiogenic medium containing MDCK cells stained green for the presence offilamentous actin (F-actin) proteins which are found in all eukaryoticcells. FIGS. 16A-16C shows the effect of the virus on the MDCK cellswithout the presence of silicon nitride.

Example 4: Effect of Silicon Nitride on Plasmopara Viticola

To show the effect of silicon nitride on the inactivation ofagricultural fungi, Cabernet Sauvignon leaves were infected withPlasmopara viticola at a concentration of 3×10⁴ spore sacs/ml. TreatedPlasmopara viticola was exposed to a slurry of 1.5 vol.% silicon nitridefor 1 minute.

FIG. 17A shows untreated Plasmopara viticola fungi on Cabernet Sauvignonleaves. FIG. 17B shows treated Plasmopara viticola fungi on CabernetSauvignon leaves. It can be seen that the leaves inoculated withPlasmopara viticola treated for 1 minute with 1.5 vol. % Si₃N₄ powderhave less of the fungi on the surface of the leaves. This is furtherevidenced by FIG. 19 which depicts the percentage of infected leaf areafor both Cabernet Sauvignon and Cannonau leaves inoculated with controland treated Plasmopara viticola. FIG. 19 clearly shows a statisticalsignificance win the infected leaf area between the control and treatedfungi.

The silicon nitride particles appear electrically attracted to andattach themselves to the spores of the pathogen, as seen in FIG. 18B.FIG. 18A shows a microscopic image of untreated spore sacs of Plasmoparaviticola, while FIG. 18B shows a microscopic image of spore sacs ofPlasmopara viticola in the presence of Si₃N₄.

Having described several embodiments, it will be recognized by thoseskilled in the art that various modifications, alternativeconstructions, and equivalents may be used without departing from thespirit of the invention. Additionally, a number of well-known processesand elements have not been described in order to avoid unnecessarilyobscuring the present invention. Accordingly, the above descriptionshould not be taken as limiting the scope of the invention.

Those skilled in the art will appreciate that the presently disclosedembodiments teach by way of example and not by limitation. Therefore,the matter contained in the above description or shown in theaccompanying drawings should be interpreted as illustrative and not in alimiting sense. The following claims are intended to cover all genericand specific features described herein, as well as all statements of thescope of the present method and system, which, as a matter of language,might be said to fall therebetween.

What is claimed is:
 1. A composition for inactivating a pathogen on aplant comprising silicon nitride at a concentration of about 1 vol. % toabout 30 vol. %.
 2. The composition of claim 1, wherein the compositioncomprises a slurry of silicon nitride particles and water.
 3. Thecomposition of claim 1, wherein the concentration of silicon nitride isabout 1.5 vol. %.
 4. The composition of claim 1, wherein theconcentration of silicon nitride is about 5 vol. % to about 30 vol. %.5. The composition of claim 1, wherein the pathogen comprises a fungi.6. The composition of claim 1, wherein the pathogen comprises Plasmoparaviticola.
 7. The composition of claim 5, wherein the plant comprisesCabernet Sauvignon or Cannonau.
 8. The composition of claim 1, whereinthe composition is suitable for treating or preventing the pathogen onthe plant.